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粟酒裂殖酵母nmt1启动子中的TATA框突变影响转录效率,但不影响转录起始点或硫胺素抑制性。

TATA box mutations in the Schizosaccharomyces pombe nmt1 promoter affect transcription efficiency but not the transcription start point or thiamine repressibility.

作者信息

Basi G, Schmid E, Maundrell K

机构信息

European Molecular Biology Laboratory, Heidelberg, Germany.

出版信息

Gene. 1993 Jan 15;123(1):131-6. doi: 10.1016/0378-1119(93)90552-e.

Abstract

The nmt1 gene of Schizosaccharomyces pombe is highly expressed and subject to transcriptional repression by thiamine. The nmt1 promoter, in common with other strong promoters in this organism, contains a canonical sequence element, 5'-ATATATAAA, located 25 bp upstream from the transcription start point (tsp). We have made stepwise deletions of the TATA box and quantitated the effects of the mutations by assaying the expression of the chloramphenicol acetyltransferase (CAT)-encoding gene (cat) cloned downstream. Our results demonstrate that progressive truncation of the TATA box results in a concomitant decrease in promoter strength as judged both by the loss of CAT activity in cell extracts and by a reduction in the steady-state level of cat mRNA. Both the induced level and the residual, repressed level of expression observed in the presence of thiamine are similarly down-regulated. On the other hand, even in the most extreme mutant, the tsp is unaffected, suggesting that other elements in the nmt1 promoter are important in determination of the tsp. The properties of the modified promoters have made them useful for extending the range of the pREP inducible expression vectors.

摘要

粟酒裂殖酵母的nmt1基因高度表达,并受到硫胺素的转录抑制。与该生物体中的其他强启动子一样,nmt1启动子包含一个典型的序列元件5'-ATATATAAA,位于转录起始点(tsp)上游25 bp处。我们逐步缺失了TATA框,并通过检测克隆在下游的氯霉素乙酰转移酶(CAT)编码基因(cat)的表达来定量突变的影响。我们的结果表明,TATA框的逐步截短导致启动子强度随之降低,这通过细胞提取物中CAT活性的丧失以及cat mRNA稳态水平的降低来判断。在硫胺素存在下观察到的诱导表达水平和残留的抑制表达水平均同样下调。另一方面,即使在最极端的突变体中,tsp也未受影响,这表明nmt1启动子中的其他元件在确定tsp方面很重要。修饰后启动子的特性使其可用于扩展pREP诱导表达载体的范围。

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